U.S. patent application number 15/309195 was filed with the patent office on 2017-03-16 for par-1 based therapeutic conjugates and uses thereof.
The applicant listed for this patent is TEL HASHOMER MEDICAL RESEARCH INFRASTRUCTURE AND SERVICES LTD.. Invention is credited to Joab CHAPMAN, Efrat SHAVIT-STEIN.
Application Number | 20170072014 15/309195 |
Document ID | / |
Family ID | 54479399 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170072014 |
Kind Code |
A1 |
CHAPMAN; Joab ; et
al. |
March 16, 2017 |
PAR-1 BASED THERAPEUTIC CONJUGATES AND USES THEREOF
Abstract
The present invention is directed to a peptide conjugate
comprising an alpha-amino protecting moiety, a peptide comprising
the amino acid sequence at least 3 amino-acid long derived from the
C'-terminus of PAR-1, or an active variant thereof and a
protease-disabling moiety. The present invention is further
directed to pharmaceutical compositions comprising the peptide
conjugate and use thereof for treating diseases and disorder
associated with excessive PAR-1 activity.
Inventors: |
CHAPMAN; Joab; (Kiryat Ono,
IL) ; SHAVIT-STEIN; Efrat; (Tel Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TEL HASHOMER MEDICAL RESEARCH INFRASTRUCTURE AND SERVICES
LTD. |
Ramat Gan |
|
IL |
|
|
Family ID: |
54479399 |
Appl. No.: |
15/309195 |
Filed: |
May 11, 2015 |
PCT Filed: |
May 11, 2015 |
PCT NO: |
PCT/IL2015/050488 |
371 Date: |
November 6, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61991532 |
May 11, 2014 |
|
|
|
62137846 |
Mar 25, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/0819 20130101;
C07K 5/0806 20130101; C07K 14/723 20130101; C07K 5/1024 20130101;
A61K 45/06 20130101; A61K 38/57 20130101; A61P 25/00 20180101; A61K
38/1796 20130101; A61K 38/00 20130101; C07K 5/101 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/57 20060101 A61K038/57; A61K 45/06 20060101
A61K045/06 |
Claims
1.-20. (canceled)
21. A peptide conjugate comprising an alpha-amino protecting
moiety, a peptide comprising the amino acid sequence at least 3
amino-acid long derived from the C'-terminus of PAR-1 as set forth
in SEQ ID NO: 17, or an active variant thereof and a
protease-disabling moiety.
22. The peptide conjugate of claim 21, wherein the alpha-amino
protecting moiety is bound to the N'-terminus amino acid sequence
of the peptide directly or via a linker.
23. The peptide conjugate of claim 21, wherein the
protease-disabling moiety is bound to the C'-terminus amino acid
sequence of the peptide directly or via a linker.
24. The peptide conjugate of claim 21, wherein the alpha-amino
protecting moiety is tosyl or derivatives thereof.
25. The peptide conjugate of claim 21, wherein the peptide
comprises the amino acid sequence Asp-Pro-Arg.
26. The peptide conjugate of claim 25, wherein the peptide
comprises an amino acid sequence as set forth in any one of SEQ ID
NO:1 to SEQ ID NO:17 and Asp-Pro-Arg, or an active variant
thereof.
27. The peptide conjugate of claim 21, wherein the
protease-disabling moiety is a substituted acetyl.
28. The peptide conjugate of claim 21, wherein the alpha-amino
protecting moiety is tosyl or a derivative thereof, the peptide
comprises the amino acid sequence Asp-Pro-Arg or an active variant
thereof and the protease-disabling moiety is a haloacetyl.
29. A pharmaceutical composition comprising a peptide conjugate of
claim 21 and a pharmaceutically acceptable carrier.
30. The pharmaceutical composition of claim 29, further comprising
trichloroacetate.
31. The pharmaceutical composition of claim 29, further comprising
a PAR-1 antagonist.
32. A method of treating a disease or disorder associated with
excessive protease receptor activity in a subject in need of such
treatment, comprising administering to said patient a
therapeutically effective amount of a pharmaceutical composition
comprising a peptide conjugate and a pharmaceutically acceptable
carrier, said peptide conjugate comprising an alpha-amino
protecting moiety, a peptide comprising the amino acid sequence at
least 3 amino-acid long derived from the C'-terminus of PAR-1 as
set forth in SEQ ID NO: 17, or an active variant thereof and a
protease-disabling moiety.
33. The method of claim 32, wherein the protease receptor is
PAR-1.
34. The method of claim 32, wherein the subject in need thereof is
a subject afflicted with said disease or disorder or a subject
susceptible to said disease or disorder.
35. The method of claim 32, wherein said disease or disorder is
selected from the group consisting of neuroinflammation,
neuroinflammatory diseases or disorders, neurodegenerative disease
or disorder, neuropathy and diabetes-related neuropathy.
36. The method of claim 32, further comprising administering a
second therapeutic agent in combination with said peptide
conjugate.
37. The method of claim 36, wherein the second therapeutic agent is
a PAR-1 antagonist.
38. A method for inducing a reduction in thrombin activity in a
subject in need of such treatment, comprising administering to said
subject a therapeutically effective amount of a pharmaceutical
composition comprising a peptide conjugate and a pharmaceutically
acceptable carrier, said peptide conjugate comprising an
alpha-amino protecting moiety, a peptide comprising the amino acid
sequence at least 3 amino-acid long derived from the C'-terminus of
PAR-1 as set forth in SEQ ID NO: 17, or an active variant thereof
and a protease-disabling moiety.
Description
RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. .sctn.371 national phase
application of PCT/IL2015/050488, filed May 11, 2015, which claims
priority to US 61/991,532 filed on May 11, 2014 and US 62/137,846
filed on Mar. 25, 2015. All applications are incorporated herein by
reference as if fully set forth.
FIELD OF THE INVENTION
[0002] The present invention is directed to a peptide conjugate
comprising an alpha-amino protecting moiety, a peptide comprising
the amino acid sequence at least 3 amino-acid long derived from the
C'-terminus of PAR-1, or an active variant thereof and a
protease-disabling moiety. The present invention is further
directed to pharmaceutical compositions comprising the peptide
conjugate and use thereof for treating diseases and disorder
associated with excessive PAR-1 activity.
BACKGROUND OF THE INVENTION
[0003] Protease activated receptors (PARs) are a family of four G
protein coupled receptors numbered PAR-1 to PAR-4. Since its
discovery in the early 90's, PAR-1 has been found in many tissues
including the brain where it is found on several cell types
including neurons, astrocytes and microglia. The first functions of
PAR-1 described in neurons and glia involved cell survival and cell
death. This dual effect is concentration dependent where at high
levels of PAR-1 activation deleterious effects are seen and at
lower levels protective effects are seen. PAR-1 carries all
determinants required for thrombin recognition.
[0004] Many neurological diseases are known to be associated with
increased inflammation and over activation of protease activated
receptors (PARs) by protein factors of the coagulation cascade.
This include, neuropathy which is commonly caused by diabetes
mellitus, occurring in 60% of all diabetic patients.
[0005] Many studies have suggested that thrombin plays a role in
angiogenesis and its inhibition reduces edema. Glioblastoma
multiform, (GBM) is a rapidly growing brain tumor resulting in high
mortality within month. Several processes characterize GBM
including increased angiogenesis, edema formation and enhanced
tumor cell proliferation.
[0006] US 2009/0281100 and US 2004/0092535 disclose a method of
inhibiting a serine/threonine kinase using benzimidazole
quinolinones, wherein the serine/threonine kinase is PAR-1, for
treating diabetes, noninsulin dependent diabetes mellitus,
Alzheimer's disease, bipolar disorder, cancer, autoimmune diseases
and organ transplant rejection.
[0007] US 2012/0232097 and US 2009/0176803 disclose
cinnamoyl-piperazine derivatives as PAR-1 antagonists and uses
thereof for treating and/or preventive arterial/venous thrombosis,
acute coronary syndromes, restenosis, stable angina, heart rhythm
disorders, myocardial infarction, hypertension, heart failure,
stroke, inflammatory disorders, pulmonary diseases,
gastrointestinal diseases, fibrosis development in chronic liver
disease patients, cancer and skin diseases.
[0008] U.S. Pat. Nos. 8,232,295 and 7,842,716 disclose methods for
treating and/or preventing vascular events by inhibiting PAR-1
and/or PAR-4 using statins.
[0009] US 2007/0142272 discloses use of an activated protein C
(APC), prodrug and/or a variant thereof as agonist of PAR-1 and/or
PAR-3 and/or endothelial protein C receptor (EPCR) for providing
neuroprotection, treating neurodegenerative disease and improving
stress or injury.
[0010] Despite efforts made in the field, there remains an unmet
need in the art for potent inhibitors that inhibit or prevent over
activation of PARs, especially, PAR-1.
SUMMARY OF THE INVENTION
[0011] The present invention provides novel peptide conjugates
capable of targeting increased protease activity associated with
diseases and disorders. The peptide conjugates of the invention are
adapted to specifically protect PAR receptors from being
over-activated. Surprisingly, the peptide conjugates of the
invention were shown to inhibit thrombin activity, reduce
thrombin-like activity generated by glioma-cells, inhibit
proliferation of glioma cells, induce reduction in glioblastoma
multiform (GBM) tumors in vivo and reduced formation of edema
associated with GBM. Advantageously, the peptide conjugates exert
the aforementioned therapeutic activities at significantly low
concentrations, within the range of nanomolars in vitro and
micromolars in vivo, while known PAR1 inhibitors require much
higher concentrations in order to exert an inhibitory activity.
Moreover, the activity of the peptide conjugate is performed
without effecting coagulation. The remarkable efficacy at nanomolar
concentrations suggests that the conjugates provide reduced
toxicity and reduced occurrence of side effects, particularly,
undesired internal bleeding.
[0012] There is provided a peptide conjugate comprising an
alpha-amino protecting moiety, a peptide comprising the amino acid
sequence at least 3 amino-acid long derived from the C'-terminus of
PAR-1 as set forth in SEQ ID NO: 17, or an active variant thereof
and a protease-disabling moiety.
[0013] In some embodiments, the alpha-amino protecting moiety may
be bound to the N'-terminus amino acid sequence of the peptide
directly or via a linker.
[0014] In some embodiments, the protease-disabling moiety may be
bound to the C'-terminus amino acid sequence of the peptide
directly or via a linker.
[0015] In some embodiments, the alpha-amino protecting moiety may
be selected from tosyl and tert-Butyloxycarbonyl.
[0016] In some embodiments, the alpha-amino protecting moiety may
be tosyl or derivatives thereof.
[0017] In some embodiments, the peptide comprises the amino acid
sequence Asp-Pro-Arg.
[0018] In some embodiments, the peptide comprises an amino acid
sequence as set forth in any one of SEQ ID NO:1 to SEQ ID NO:17 and
Asp-Pro-Arg, or an active variant thereof.
[0019] In some embodiments, the peptide comprises the amino acid
set forth in SEQ ID NO:3, or an active variant thereof.
[0020] In some embodiments, the peptide comprises the amino acid
set forth in SEQ ID NO:4, or an active variant thereof.
[0021] In some embodiments, the peptide moiety may consist of an
amino acid sequence selected from the group consisting of
Asp-Pro-Arg and SEQ ID NO: 1 to SEQ ID NO:17.
[0022] In some embodiments, the linker may be a peptide linker or
an oligonucleotide linker.
[0023] In some embodiments, the protease-disabling moiety may be a
substituted acetyl.
[0024] In some embodiments, the protease-disabling moiety may be
selected from the group consisting of sulfonylfluorides,
chloromethylketones, esters, boronic acids, aldehydes, arylketones,
trifluoromethylketones, ketocarboxylic acids and combinations
thereof.
[0025] In some embodiments, the protease-disabling moiety may be
chloromethylketone and derivatives thereof.
[0026] In some embodiments, the alpha-amino protecting moiety may
be tosyl or a derivative thereof, the peptide comprises the amino
acid sequence Asp-Pro-Arg or an active variant thereof and the
protease-disabling moiety may be a haloacetyl.
[0027] There is provided a pharmaceutical composition comprising a
peptide conjugate and a pharmaceutically acceptable carrier, said
peptide conjugate comprising an alpha-amino protecting moiety, a
peptide comprising the amino acid sequence at least 3 amino-acid
long derived from the C'-terminus of PAR-1 as set forth in SEQ ID
NO: 17, or an active variant thereof and a protease-disabling
moiety.
[0028] In some embodiments, the pharmaceutical composition may
further include trichloroacetate.
[0029] In some embodiments, the pharmaceutical composition may
further include a PAR-1 antagonist.
[0030] In some embodiments, the PAR-1 antagonist may be SCH79797
(N3-Cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3,2-f]quin-
azoline-1,3-diamine dihydrochloride).
[0031] There is provided use of a pharmaceutical composition
comprising a peptide conjugate and a pharmaceutically acceptable
carrier, said peptide conjugate comprising an alpha-amino
protecting moiety, a peptide comprising the amino acid sequence at
least 3 amino-acid long derived from the C'-terminus of PAR-1 as
set forth in SEQ ID NO: 17, or an active variant thereof and a
protease-disabling moiety, for the treatment of a disease or
disorder associated with excessive protease receptor activity.
[0032] In some embodiments, the protease receptor may be PAR-1.
[0033] In some embodiments, the disease or disorder may be selected
from the group consisting of neuroinflammation, neuroinflammatory
diseases or disorders, neurodegenerative disease or disorder,
neuropathy and diabetes-related neuropathy.
[0034] In some embodiments, the use may be for the treatment of a
tumor associated with said disease or disorder.
[0035] In some embodiments, the diseases or disorders may be
selected from the group consisting of: glioma, astrocytomas,
cancer, solid tumor, brain tumor, glioblastoma, oligodendroglioma,
ependymoma, mixed gliomas glioblastoma multiforme, neuropathy,
diabetic peripheral neuropathy, Guillain-Barre syndrome,
amyotrophic lateral sclerosis, acute inflammatory demyelinating
polyneuropathy, acute disseminated encephalomyelitis, optic
neuritis, transverse myelitis, neuromyelitis optica, Alzheimer's
disease, Huntington's disease, amyotrophic lateral sclerosis,
epilepsy, multiple sclerosis and Parkinson's disease.
[0036] In some embodiments, the treatment may include any one or
more of inhibition of the progression of said disease or disorder,
attenuation of the progression of said disease or disorder or
prevention deterioration of said disease or disorder.
[0037] There is provided a method of treating a disease or disorder
associated with excessive protease receptor activity in a patient
in need of such treatment, the method comprises the step of
administering to said patient a therapeutically effective amount of
a pharmaceutical composition comprising a peptide conjugate and a
pharmaceutically acceptable carrier, said peptide conjugate
comprising an alpha-amino protecting moiety, a peptide comprising
the amino acid sequence at least 3 amino-acid long derived from the
C'-terminus of PAR-1 as set forth in SEQ ID NO: 17, or an active
variant thereof and a protease-disabling moiety.
[0038] In some embodiments, the pharmaceutical composition may be
administered topically, intraperitoneally, systemically or
intra-cranially.
[0039] In some embodiments, the treating may include any one or
more of inhibiting the progression of said disease or disorder,
attenuating the progression of said disease or disorder or
preventing deterioration of said disease or disorder.
[0040] In some embodiments, the subject in need thereof may be a
subject afflicted with said disease or disorder or a subject
susceptible to said disease or disorder.
[0041] In some embodiments, the method may further comprise
administering a second therapeutic agent in combination with said
peptide conjugate.
[0042] In some embodiments, the second therapeutic agent may be a
PAR-1 antagonist.
[0043] There is provided use of a pharmaceutical composition
comprising a peptide conjugate and a pharmaceutically acceptable
carrier, said peptide conjugate comprising an alpha-amino
protecting moiety, a peptide comprising the amino acid sequence at
least 3 amino-acid long derived from the C'-terminus of PAR-1 as
set forth in SEQ ID NO: 17, or an active variant thereof and a
protease-disabling moiety, for inducing a reduction in thrombin
activity.
[0044] There is provided a kit for treating a disease or disorder
associated with excessive protease receptor activity in a patient
in need of such treatment, the kit comprises at least one container
comprising a therapeutically effective amount of a pharmaceutical
composition comprising a peptide conjugate and a pharmaceutically
acceptable carrier, said peptide conjugate comprising an
alpha-amino protecting moiety, a peptide comprising the amino acid
sequence at least 3 amino-acid long derived from the C'-terminus of
PAR-1 as set forth in SEQ ID NO: 17, or an active variant thereof
and a protease-disabling moiety.
[0045] In some embodiments, the kit may further comprise at least
one second container that includes a second therapeutic agent. In
some embodiments, the second therapeutic agent may be a PAR-1
antagonist.
[0046] In some embodiments, the kit may further include
instructions for use of the pharmaceutical composition for the
treatment of the disease or disorder. In some embodiments, the
instructions for use may include instructions for using the
pharmaceutical composition in the at least one first container in
combination with the pharmaceutical composition in the at least one
second container. In some embodiments, the instructions may further
recite a web site address providing further information, and
troubleshooting among other services. In some embodiments, the
instructions may be on paper form such as a package insert or
label.
[0047] Further embodiments and the full scope of applicability of
the present invention will become apparent from the detailed
description given hereinafter. However, it should be understood
that the detailed description and specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows mean thrombin-like activity (U/ml.+-.SEM) in
sciatic nerves of (A) EAN rats at day of induction and at 10, 14
and 19 days post induction (EAN 10, n=10; EAN14, n=10; EAN19, n=10,
respectively) vs. control rats (n=10) and CFA (complete Freund's
adjuvant) mice; and in (B) STZ rats (n=15) vs. control rats (n=12)
(**=p<0.01; *=p<0.005).
[0049] FIG. 2 shows Western blots (A, C) and the corresponding
statistical analyses (B, D) from 3 independent experiments of PAR-1
immunoreactivity (A, B) and PN-1 immunoreactivity (C, D) in
desheathed sciatic nerve from STZ-induced diabetic rats (n=3) and
control non-diabetic rats (n=3; *=p<0.05; **=p<0.01).
[0050] FIG. 3 presents the effect of (i) N alpha-tosyl-L-lysine
chloromethyl ketone (TLCK, 4.4 mg/kg, n=5) and (ii)
Na-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidine
(NAPAP, NAPAP-120 .mu.M/kg body weight, n=5), treatments (black
bars) relative to control/CFA (grey bars, n=3) and relative to EAN
untreated rats (EAN, n=5) through electrophysiological tests:
latency (A), conduction velocity (B) and amplitude (C). Statistics
are indicated as follows: *=p<0.05, **=p<0.01.
[0051] FIG. 4 shows the effect of N alpha-tosyl-L-lysine
chloromethyl ketone (TLCK, 4.4 mg/kg body weight, n=8) and (ii)
Na-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidine
(NAPAP, 120 .mu.M/kg body weight, n=5), treatments (black bars)
relative to control (grey bars, n=4) and STZ-induced diabetic
untreated rats (n=5) through electrophysiological tests: latency
(A), conduction velocity (B) and amplitude (C). *=p<0.05,
**=p<0.01.
[0052] FIG. 5 exhibits Western blots of PAR-1 and actin (A) and a
corresponding statistical analysis (B) in brain homogenates derived
from SOD-1 and wild-type control mice (p<0.001).
[0053] FIG. 6 exhibits a Kaplan-Meier survival curve of (full
diamond) untreated (control) SOD-1 mice, (empty circle) SOD-1 mice
treated with TLCK (4.4 mg/kg), and (empty triangle) mice treated
with PAR-1 antagonist (SCH79797; 25 .mu.As/kg).
[0054] FIG. 7 exhibits the Thrombin-like activity before (black
bars) and after treatment with NAPAP (grey bars) in C6 (A) and
CNS-1 (B) glioma cells and their surrounding medium.
[0055] FIG. 8A exhibits proliferation of C6 control cells (black
bar) and cells treated with various doses of PAR-1 antagonist
SCH79797 (grey bars).
[0056] FIG. 8B shows proliferation of CNS-1 control cells (black
bar) and cells treated with each of PAR-1 antagonists: SCH79797,
NAPAP and TLCK, and combinations thereof (grey bars).
[0057] FIG. 9 exhibits proliferation of CNS-1 glioma cell-line upon
dose-dependent treatment with TLCK (grey bars) compared to
non-treated cells (black bar).
[0058] FIG. 10 presents the effect of the various doses of the
peptide conjugates encompassing the peptide Asp-Pro-Arg (short
peptide; 3AA) and the peptide set forth in SEQ ID NO: 4 (long
peptide; 7AA) on proliferation of CNS-1 glioma cell-line (grey
bars) relative to control (non-treated cells black bar).
[0059] FIG. 11 presents dose-dependent inhibition of glioma
proliferation by different concentrations of the peptide conjugates
encompassing the peptide Asp-Pro-Arg and the peptides of SEQ ID
NOs: 2-4 (11A-11D, respectively) after 48 h.
[0060] FIG. 12 presents dose-dependent inhibition of glioma
proliferation by different concentrations of the peptide conjugates
encompassing the peptide Asp-Pro-Arg and the peptides of SEQ ID
NOs: 2-4 (12A-12D, respectively) after 72 h.
[0061] FIG. 13 presents the effect of the peptide conjugates
encompassing the peptide set forth in SEQ ID NO: 4 (7AA) with or
without PAR-1 antagonist (1 .mu.M) on proliferation (XTT) of CNS-1
glioma cell-line (grey bars) compared to untreated cells (control;
black bar).
[0062] FIG. 14 presents thrombin-like activity in thrombin
inhibition assay under various doses of the peptide conjugates
(from 156 nanomolar to 1mM to) encompassing the peptide Asp-Pro-Arg
(3AA; A) and the peptide set forth in SEQ ID NO: 4 (7AA; B)
relative to control (i.e. without the presence of an of the peptide
conjugates (diamonds).
[0063] FIG. 15 presents thrombin activity (ratio) performed in a
thrombin activity assay (non-cellular) under different
concentrations of the peptide conjugates encompassing the peptide
Asp-Pro-Arg (3AA; x, grey line) or the peptides set forth in SEQ ID
NO: 1 (4AA; x, dark line); SEQ ID NO: 2 (5AA, triangle, dark line);
SEQ ID NO: 3 (6AA; square, grey line) and SEQ ID NO: 4 (7AA;
diamond, dark line).
[0064] FIG. 16 presents the effect of the various doses of the
peptide conjugates encompassing the peptide Asp-Pro-Arg (3AA) and
the peptide set forth SEQ ID NO: 4 (7AA) on thrombin-like activity
in CNS-1 glioma cell-line (grey bars) compared to untreated cells
(control; black bar).
[0065] FIG. 17 presents thrombin activity (ratio) generated by rat
glioma cells inhibited by different concentrations of the peptide
conjugates encompassing the peptide Asp-Pro-Arg (3AA; asterisk) or
the peptides set forth in SEQ ID NO: 1 (4AA; x); SEQ ID NO: 2 (5AA;
triangle); SEQ ID NO: 3 (6AA; square) and SEQ ID NO: 4 (7AA;
diamond).
[0066] FIG. 18 presents thrombin activity in plasma samples after
addition of the peptide conjugates encompassing the peptide
Asp-Pro-Arg (A) or the peptides set forth in SEQ ID NO: 2 (B); SEQ
ID NO: 3 (C); and SEQ ID NO: 4 (D).
[0067] FIG. 19 presents sciatic nerve conduction velocity (m/s) in
a Diabetes melitus mice model (induced by streptozotocin (STZ)
injection) untreated (STZ) or treated with T5AACK (a peptide
conjugate encompassing the peptide 6AA (SEQ ID NO: 2) nested
between tosyl and CK) at different concentrations and compared to
normal mice without diabetes (Control).
[0068] FIG. 20 presents changes in tumor size (A) and in the size
of edema surrounding the tumor (B) in 3 groups of rats: (1)
control; (2) treatment with 2 .mu.M of a peptide conjugate
comprising the peptide termed 6AA (SEQ ID NO: 3); and (3) treatment
with 20 .mu.M of a peptide conjugate comprising the peptide termed
6AA (SEQ ID NO: 3).
[0069] FIG. 21 presents the percentage survival of rats in the
following treatment groups: control (saline; circle), 2 .mu.M of a
peptide conjugate comprising the peptide termed 6AA (SEQ ID NO: 3;
square), and 20 .mu.M of a peptide conjugate comprising the peptide
termed 6AA (SEQ ID NO: 3; diamond).
DETAILED DESCRIPTION OF THE INVENTION
[0070] The present invention provides novel peptide conjugates that
target an increased protease activity associated with diseases and
disorders by specifically protecting PAR receptors from being
over-activated.
[0071] It has long been recognized that in PAR-1 (NP_001983.2) the
extracellular fragment (amino-terminal exodomain) carries all
determinants for thrombin recognition. In some embodiments, the
sequence of said amino-terminal exodomain is as follows:
.sup.38LDPRSFLLRNPNDKYEPF.sup.55 (SEQ ID NO: 18). This peptide
sequence is bridging thrombin active site and exosite I.
Furthermore, the sequence .sup.38LDPR.sup.41 (also termed LDPR; SEQ
ID NO: 1), is contacting the thrombin active site and the acidic
hirudin-like motif, .sup.51KYEPF.sup.55 (SEQ ID NO: 19), thereby
engaging thrombin exosite I. Although the highest binding affinity
and maximal proximity was found between .sup.38LDPR.sup.41 (P1 to
P4; (SEQ ID NO: 1) and the active site of thrombin, a 3D modeling
of PAR-1 N-terminal peptide and thrombin reveals contacts (with
lower strength) up to amino acid at position 20 (P20). PAR-1
activation requires specific binding of thrombin and similar
proteases to a binding site on the receptor, which is just
preceding the cleavage site. The binding site on the receptor PAR-1
comprises the amino acid sequence PESKATNATLDPR (SEQ ID NO: 10)
that is significantly different from the equivalent sequences in
PAR-2, 3 and 4. As exemplified in Table 2, hereinbelow, there is a
high degree of homology between the human, mouse, rat and bovine
PAR-1 in positions P1 to P7, wherein the highest homology is at
P1-P2 and P5-P7.
[0072] In the nervous system a major factor is played by the
activation of PARs on glia cells. In the peripheral nervous system
(PNS), PAR-1 is localized to the non-compacted myelin of Schwann
cells at the node of Ranvier, and in the neuromuscular junction it
is localized to the peri-synaptic glia and the post-synaptic
muscle. The inventors of the present invention were the first to
show a functional role for PAR-1 activation on the glia component
of the node of Ranvier in the PNS where activation of the receptor
caused nerve conduction block (Shavit et al., Brain, 2008, vol.
131(Pt 4): p. 1113-1122). In the central nervous system (CNS) PAR-1
was found on neurons and glia. It was suggested that PAR-1 is
localized to a specific astrocyte structure at the synapse, and the
inventors of the present invention have confirmed this location by
high resolution methods such as confocal and electron microscopy
(Shavit et al., J Neurochem. 119(3): p. 460-473). PAR-1 activation
was shown to hold a physiological role at the CNS synapse where its
activation modulates synaptic transmission by causing long-term
potentiation (LTP) and seizure-like activity and potentiates the
synaptic N-methyl-D-aspartate (NMDA) receptor. It is interesting to
note therefore that PAR-1 is a marker of glial structures adjacent
to the most physiologically active parts of the nervous system, the
synapse and the node of Ranvier.
[0073] Thus, there is provided a peptide conjugate comprising a
protecting moiety, a peptide comprising the amino acid sequence DPR
(also termed Asp-Pro-Arg) or an active variant thereof and a
thrombin-disabling moiety.
[0074] In some embodiments, the peptide conjugate may comprise the
following structure: PRO-PEP-DIS, wherein PRO is a protecting
moiety, such as, an alpha-amino protecting moiety; PEP is a peptide
moiety comprising the amino acid sequences DPR or amino acid
sequences comprising same, as set forth in SEQ ID NO:2 to SEQ ID
NO:17, or an active variant thereof; and DIS may be a
protease-disabling moiety, wherein PRO may be bound to the N'
-terminus amino acid of PEP directly or via a linker, and wherein
DIS may be bound to the C'-terminus amino-acid of PEP directly or
via a linker. Each possibility represents a separate embodiment of
the present invention.
[0075] The terms "peptide moiety" and "PEP" are interchangeable and
refer to a peptide moiety derived from the PAR-1, including, but
not limited to, the C'-terminus of PAR-1 (SEQ ID NO: 17).
[0076] In some embodiments, the peptide moiety may have an
amino-acid sequence at least 3 amino acid long derived from the
C'-terminus of PAR-1.
[0077] In some embodiments, the peptide moiety may have an
amino-acid sequence at least 3 amino-acid long and at most 7
amino-acid long, derived from the C'-terminus of PAR-1.
[0078] In some embodiments, the peptide moiety may have an
amino-acid sequence of at least 3 amino-acid long and at most 7
amino-acid long derived from the C'-terminus of PAR-1 starting from
Arginine at position 20 and continuing towards the N'
-terminus.
[0079] In some embodiments, the amino-acid sequences of DPR and SEQ
ID NO: 1 to SEQ ID NO:17 are fragments of the PAR-1 receptor, to
which thrombin binds and the peptide conjugates of the present
invention comprise such sequence as decoy for thrombin i.e. to
enable their specific binding to thrombin.
[0080] In some embodiments, the peptide moiety comprises an
amino-acid sequence selected from the group consisting of DPR, LDPR
(SEQ ID NO: 1), TLDPR (SEQ ID NO: 2), ATLDPR (SEQ ID NO: 3),
NATLDPR (SEQ ID NO: 4), TNATLDPR (SEQ ID NO: 5), ATNATLDPR (SEQ ID
NO: 6), KATNATLDPR (SEQ ID NO: 7), SKATNATLDPR (SEQ ID NO: 8),
ESKATNATLDPR (SEQ ID NO: 9), PESKATNATLDPR (SEQ ID NO: 10),
RPESKATNATLDPR (SEQ ID NO: 11), RRPESKATNATLDPR (SEQ ID NO: 12),
ARRPESKATNATLDPR (SEQ ID NO: 13), RARRPESKATNATLDPR (SEQ ID NO:
14), TRARRPESKATNATLDPR (SEQ ID NO: 15), RTRARRPESKATNATLDPR (SEQ
ID NO: 16) and ARTRARRPESKATNATLDPR (SEQ ID NO: 17). Each
possibility represents a separate embodiment of the present
invention.
[0081] In some embodiments, the peptide moiety may consist of
Asp-Pro-Arg (DPR). In some embodiments, the peptide moiety may
consist of SEQ ID NO: 1. In some embodiments, the peptide moiety
may consist of SEQ ID NO: 2. In some embodiments, the peptide
moiety may consist of SEQ ID NO: 3. In some embodiments, the
peptide moiety may consist of SEQ ID NO: 4. In some embodiments,
the peptide moiety may consist of SEQ ID NO: 5. In some
embodiments, the peptide moiety may consist of SEQ ID NO: 6. In
some embodiments, the peptide moiety may consist of SEQ ID NO: 7.
In some embodiments, the peptide moiety may consist of SEQ ID NO:
8. In some embodiments, the peptide moiety may consist of SEQ ID
NO: 9. In some embodiments, the peptide moiety may consist of SEQ
ID NO: 10. In some embodiments, the peptide moiety may consist of
SEQ ID NO: 11. In some embodiments, the peptide moiety may consist
of SEQ ID NO: 12. In some embodiments, the peptide moiety may
consist of SEQ ID NO: 13. In some embodiments, the peptide moiety
may consist of SEQ ID NO: 14. In some embodiments, the peptide
moiety may consist of SEQ ID NO: 15. In some embodiments, the
peptide moiety may consist of SEQ ID NO: 16. In some embodiments,
the peptide moiety may consist of SEQ ID NO: 17.
[0082] In some embodiments, the peptide moiety may comprise an
amino-acid sequence Asp-Pro-Arg or an active variant thereof In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 1 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 2 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 3 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 4 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 5 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 6 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 7 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 8 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 9 or an active variant thereof. In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 10 or an active variant thereof In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 11 or an active variant thereof.
In some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 12 or an active variant thereof In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 13 or an active variant thereof.
In some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 14 or an active variant thereof In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 15 or an active variant thereof.
In some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 16 or an active variant thereof In
some embodiments, the peptide moiety may comprise an amino-acid
sequence set forth in SEQ ID NO: 17 or an active variant
thereof.
[0083] The terms "active variant", "analogue" and "variant" as used
herein are interchangeable and refer to any peptide moiety derived
from a peptide sequence as set forth in DPR and any one of SEQ ID
NO:1 to SEQ ID NO:17 by at least one amino-acid substitution, that
retains at least 70%, optionally, at least 80% or at least 90% or
at least 95%, of the biological activity of the sequence from which
it was derived, or to which it is most similar to. These terms also
encompass peptides comprising regions having substantial similarity
to the peptide moiety, such as structural variants.
[0084] The term "substantial similarity" means that two peptide
sequences, when optimally aligned, share at least 50 percent
sequence identity, at least 60 percent sequence identity, at least
70 percent sequence identity, at least 80 percent sequence
identity, at least 90 percent sequence identity, or at least 95
percent sequence identity or more (e.g., 99 percent sequence
identity). Typically, residue positions, which are not identical,
differ by conservative amino acid substitutions.
[0085] In some embodiments, one or more of the peptide moieties may
correspond to variants of the amino-acid sequence DPR or the amino
acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:17. Each
possibility represents a separate embodiment of the present
invention.
[0086] In some embodiments, said variants may comprise conservative
substitutions relative to the amino acid sequence of the peptide
moiety corresponding thereto.
[0087] Examples of conservative substitutions as considered in the
present invention are the substitution of any positive-charge
amino-acid (Arg, His, Lys) with any other positive-charge
amino-acid; the substitution of any negative-charge amino-acid
(Asp, Glu) with any other negative-charge amino-acid; the
substitution of any polar-uncharged amino-acid (Ser, Thr, Asn, Gln)
with any other polar-uncharged amino-acid; or the substitution of
any hydrophobic amino-acid (Ala, Ile, Leu, Met, Phe, Trp, Tyr, Val)
with any other hydrophobic amino-acid.
[0088] Thus, in some embodiments, active variant may comprise
Arg/His/Lys substitution; Asp/Glu substitution; Ser/Thr/Asn/Gln
substitution; Ala/Ile/Leu/Met/Phe/Trp/Tyr/Val substitution; or any
combination of the above. Each possibility represents a separate
embodiment of the present invention.
[0089] In some embodiments, the peptide may be selected from the
amino-acid sequences DPR and those set forth in SEQ ID NOs: 1 to
17, wherein at least on proline is substituted with a
positive-charge amino acid. In other embodiments, the peptide is
selected from DPR and SEQ ID NOs: 1 to 17, wherein at least on
proline is substituted with lysine. Without being bound by any
theory of mechanism, the peptide is substituted in order to obtain
improved specificity to thrombin and potentially other coagulation
factors, improved penetration into the brain and prolonged
half-life of the conjugate.
[0090] Residue positions, which are not identical, may also be
composed of peptide analogs, including unnatural amino acids or
derivatives of such. Analogs typically differ from naturally
occurring peptides at one, two or a few positions, often by virtue
of conservative substitutions.
[0091] Some analogs may also include unnatural amino acids or
modifications of N or C terminal amino acids at one, two or a few
positions. Examples of unnatural amino acids, without limiting to,
are D-amino acids, alpha, alpha-disubstituted amino acids, N-alkyl
amino acids, lactic acid, 4-hydroxyproline, y-carboxyglutamate,
epsilon-N,N,N-tri methyllysine, epsilon-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, omega-N-methylarginine, and
isoaspartic acid.
[0092] The terms "protecting moiety" and "PRO" are interchangeable
and refer to any moiety capable of protecting the peptide conjugate
of the present invention from adverse effects such as proteolysis,
degradation or clearance, or alleviating such adverse effects.
[0093] In some embodiments, the protecting moiety may be an
alpha-amino protecting moiety. In some embodiments, the protecting
moiety may be tosyl (a tosyl group) or derivatives thereof.
[0094] In some embodiments, the alpha-amino protecting moiety may
be tosyl.
[0095] In some embodiments the protecting moiety is selected from
the group consisting of t-butyloxycarbonyl (BOC,
(CH.sub.3).sub.3COCO-, t-BOC), t-amyloxycarbonyl,
adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl,
9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and
the like, nitro, tosyl (CH.sub.3C.sub.6H.sub.4SO.sub.2--),
benzyloxycarbonyl (CBZ), adamantyloxycarbonyl,
2,2,5,7,8-pentamethylchroman-6-sulfonyl,
2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or
substituted BZL, such as, p-methoxybenzyl, p-nitrobenzyl,
p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl. Each
possibility represents a separate embodiment of the present
invention.
[0096] In some embodiments, PRO may be selected from t-butyl,
cyclohexyl, cyclopentyl, benzyloxymethyl (BOM), tetrahydropyranyl,
trityl, chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl. Each
possibility represents a separate embodiment of the present
invention.
[0097] Other protecting groups which may suitably employed are
bromobenzyloxycarbonyl, xanthyl (Xan) and p-methoxybenzyl. Each
possibility represents a separate embodiment of the present
invention.
[0098] In some embodiments, PRO may be tosyl.
[0099] The terms "protease-disabling moiety" and "DIS" as used
herein are interchangeable and refer to any moiety capable of
binding to a protease and transiently or permanently disabling its
proteolytic activity. In some embodiments, the protease-disabling
moiety may be a thrombin-disabling moiety. In some embodiments, the
protease disabling moiety may be a thrombin inhibitor.
[0100] In some embodiments, the protease-disabling moiety may be a
protease-disabling compound selected from irreversible inhibitors
and reversible inhibitors.
[0101] In some embodiments, the protease-disabling moiety may be an
irreversible inhibitor selected from the group consisting of
substituted acetyl (1-x-actyl), sulfonylfluorides (--SO.sub.2F),
chloromethylketones (--COCH.sub.2Cl), esters (--COOR), boronic
acids (--B(OR).sub.2) and combinations thereof.
[0102] In some embodiments, the protease-disabling moiety may be a
reversible inhibitor selected from the group consisting of
aldehydes (--CHO), arylketones (--CO-Aryl), trifluoromethylketones
(--COCF.sub.3) ketocarboxylic acids (--COCOOH) and combinations
thereof.
[0103] In some embodiments the protease-disabling moiety may be a
protease-disabling compound selected from the group consisting of
chloromethylketone (CK) and derivatives thereof, sulfonylfluorides
(--SO.sub.2F), chloromethylketones (--COCH.sub.2Cl), esters
(--COOR), boronic acids (--B(OR).sub.2), aldehydes (--CHO),
arylketones (--CO-Aryl), trifluoromethylketones (--COCF.sub.3) and
ketocarboxylic acids (--COCOOH).
[0104] In some embodiments, the protease-disabling moiety may be a
substituted acetyl. In some embodiments, the substituted acetyl may
be haloacetyl. In some embodiments, the haloacetyl may be
chloroacetyl. In some embodiments, the protease-disabling moiety
may be chloromethylketone (CK).
[0105] In some embodiments, the peptide conjugate may be
tosyl-DPR-CK. In some embodiments, the peptide conjugate may be
tosyl-LDPR-CK. In some embodiments, the peptide conjugate may be
tosyl-TLDPR-CK. In some embodiments, the peptide conjugate may be
tosyl-ATLDPR-CK. In some embodiments, the peptide conjugate may be
tosyl-NATLDPR-CK.
[0106] In some embodiments, the peptide conjugate may be
tosyl-PEP-CK, wherein PEP is Aspartic acid-Proline-Arginine or any
one of the peptides set forth in SEQ ID NOs: 1-17, or an active
variant thereof, wherein CK may be bound to the C'-terminus
amino-acid of Arginine and tosyl may be bound to the N' -terminus
amino acid of PEP.
[0107] Several thrombin inhibitors are known in the art, including
T-L-C-K (also known as N alpha-tosyl-L-lysine chloromethyl ketone
or TLCK), NAPAP (also known as
Na-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidine),
PN-1 (also known as Protease nexin-1), PN-2 (also known as Protease
nexin-2, APP) and SCH79797 (also known as
N3-Cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3,2-f]
quinazoline-1,3 -diamine dihydrochloride). TLCK is an irreversible
inhibitor of the serine protease trypsin (inactivates trypsin most
rapidly at pH 7.5), and many trypsin-like serine proteases. The
histidine-46 residue located in the active site of trypsin is
alkylated by TLCK. NAPAP binds thrombin in the S1, S2 and S4
pockets. The amidine group on NAPAP forms a bidentate salt-bridge
with Asp deep in the S1 pocket, the piperidine group takes the role
of proline residue and binds in the S2 pocket, and the naphthyl
rings of the molecule forms a hydrophobic interaction with Trp in
the S4 pocket. PN-1 is a 43 kDa thrombin inhibitor, member of the
serine protease inhibitor superfamily (serpins), which regulates
matrix accumulation and coagulation under pathophysiologic
conditions by inhibiting thrombin, plasmin, and tissue plasminogen
activators. PN-2 is a protease inhibitor, which is the secreted
form of the amyloid beta-protein precursor (APP) which contains a
Kunitz protease inhibitor domain. SCH79797 is a potent and
selective non-peptide antagonist of protease activated receptor-1
(PAR-1).
[0108] However, the alleged therapeutic effect of the known
inhibitors is accompanied by serious adverse effects. For example,
as detailed below, the very short peptide T-L-C-K, induced
inhibition of glioma-cell proliferation, at relatively high
concentrations (mM) but caused severe internal bleeding.
[0109] Surprisingly, the peptide conjugates of the invention, which
are based on the thrombin binding site on PAR-1, provide the
required therapeutic effect without causing mortal side effect. As
exemplified hereinbelow, the peptide conjugates were shown to
effectively inhibit glioma growth and thrombin activity.
[0110] In some embodiments, the N' terminal amino acid of PEP may
covalently bind to PRO, directly or via a linker, through its N'
amino-group or through its side-chain. In some embodiments, the C'
terminal amino acid of PEP may covalently bind to DIS, directly or
via a linker, through its C' carboxyl-group or through its
side-chain.
[0111] In some embodiments, PRO may be bound to PEP via a
linker.
[0112] The term "linker" as used herein refers to any molecule
bound to both PRO and PEP and/or to both PEP and DIS. In its
simplest form, a linker may be a covalent bond. More elaborate
linkers may be amino-acid moieties, peptide moieties, nucleotide
moieties, oligonucleotide moieties etc. Contemplated linkers may
also serve a further therapeutic purpose, for example, they may be
fluorescent, thereby enabling detection of the peptide conjugates
carrying them, or they may be a polyethylene glycol (PEG) moiety,
further protecting the peptide conjugates carrying them from
degradation. Thus, in some embodiments, said linker may be selected
from the group consisting of a peptide linker and an
oligonucleotide linker. Each possibility represents a separate
embodiment of the present invention.
[0113] In some embodiments, there is provided a pharmaceutical
composition, comprising the peptide conjugate described above, as a
pharmaceutically active ingredient, and a pharmaceutically
acceptable carrier. In some embodiments, the pharmaceutical
composition may further comprises trichloroacetate (TCA) salt.
[0114] The term "pharmaceutically acceptable carrier" as used
herein refers to any of the standard pharmaceutical carriers known
in the field such as sterile solutions, tablets, coated tablets,
and capsules. Typically, such carriers contain excipients such as
starch, milk, sugar, certain types of clay, gelatin, stearic acids
or salts thereof, magnesium or calcium stearate, talc, vegetable
fats or oils, gums, glycols, or other known excipients. Such
carriers may also include flavor and color additives or other
ingredients.
[0115] Examples of pharmaceutically acceptable carriers include,
but are not limited to, the following: water, saline, buffers,
inert, nontoxic solids (e.g., mannitol, talc).
[0116] Compositions comprising such carriers are formulated by
well-known conventional methods. Depending on the intended mode of
administration and the intended use, the compositions may be in the
form of solid, semi-solid, or liquid dosage forms, such, for
example, as powders, granules, crystals, liquids, suspensions,
liposomes, nano-particles, nano-emulsions, pastes, creams, salves,
etc., and may be in unit-dosage forms suitable for administration
of relatively precise dosages.
[0117] In some embodiments, the pharmaceutical composition of the
invention may be formulated for oral, nasal, aerosol, inhalational,
abdominal, subcutaneous, intra-peritoneal or intravenous
administration. Each possibility represents a separate embodiment
of the present invention.
[0118] In some embodiments, the pharmaceutical composition of the
invention may be for intravenous administration.
[0119] In some embodiments, there is provided use of a peptide
conjugate as described above, for the treatment of a disease or
disorder associated with excessive protease activity.
[0120] In some embodiments, the use of the peptide conjugate may
further include use of a second therapeutic agent. In some
embodiments, the second therapeutic agent may be a PAR-1
antagonist.
[0121] As used herein, the term `excessive protease activity`
refers to activity which is at least twice as high as normal
activity of the protease. In some embodiments, excessive protease
activity refers to an activity that is at least three times higher
the normal activity of the protease. For example, typical excess
activity of protease in stroke, diabetes, ALS, EAE, trauma and
glioma is two to three times higher than the protease activity in
control subjects. In another example, the excess activity in stroke
is up to 100 times higher than the protease activity in control
subjects.
[0122] The term "disease or disorder associated with excessive
protease activity" as used herein refers to any disease or disorder
known to be caused and/or manifested by a higher than normal
protease activity, as determined by standard methods well known in
the art.
[0123] In some embodiments, there is provided a method for treating
a disease or disorder associated with excessive protease receptor
activity comprising administering to a subject in need thereof, a
pharmaceutical composition comprising a peptide conjugate according
to the present invention and a carrier,
[0124] In some embodiments, said disease or disorder may be
selected from the group consisting of neuroinflammation,
neuroinflammatory diseases or disorders, neurodegenerative disease
or disorder, neuropathy, and diabetes-related neuropathy. Each
possibility represents a separate embodiment of the present
invention.
[0125] Diabetic peripheral neuropathy (DPN), one of the most
prevalent forms of diabetes-related neuropathy, principally
manifests itself as sensory loss and weakness of the lower limbs
and ultimately accounts for significant morbidity contributing to
amputation. In the PNS the prototypical inflammatory disease
affecting nerve cells is the Guillain-Barre syndrome (GBS). GBS is
a group of acute inflammatory neuropathies and its animal model is
experimental autoimmune neuritis (EAN). Acute inflammatory
demyelinating polyneuropathy (AIDP) is the most common form of GBS,
and is characterized by demyelination of peripheral axons and
axonal degeneration in severe cases. The mechanism leading to GBS
are still being elucidated and most of the evidence points to an
immune attack at the node of Ranivier including the glial
components. Amyotrophic lateral sclerosis (ALS) also affects
peripheral nerve by causing selective degeneration of lower motor
nerves together with degeneration of upper motor neurons in the
brain. The cause of this disease is not known and treatment for it
is limited to the use of riluzol, a glutamate receptor antagonist,
which is only partially effective and patients with this disease
will die within 2 years of diagnosis.
[0126] In some embodiments, said diseases or disorders may be
selected from the group consisting of: glioma, astrocytomas,
glioblastoma, oligodendroglioma, ependymoma, mixed gliomas
glioblastoma multiforme, neuropathy, diabetic peripheral
neuropathy, Guillain-Barre syndrome, amyotrophic lateral sclerosis,
acute inflammatory demyelinating polyneuropathy, acute disseminated
encephalomyelitis, optic neuritis, transverse myelitis,
neuromyelitis optica, Alzheimer's disease, Huntington's disease,
amyotrophic lateral sclerosis, epilepsy, multiple sclerosis and
Parkinson's disease. Each possibility represents a separate
embodiment of the present invention.
[0127] In some embodiments, treating may include any one or more of
inhibiting the progression of said disease or disorder, attenuating
the progression of said disease or disorder or preventing
deterioration of said disease or disorder. Each possibility
represents a separate embodiment of the present invention.
[0128] In some embodiments, there is provided use of a peptide
conjugate described above, for the preparation of a medicament for
the treatment of a disease or disorder associated with excessive
protease receptor activity. Each possibility represents a separate
embodiment of the present invention.
[0129] In some embodiments, there is provided a method of treating
a disease or disorder associated with excessive protease receptor
activity in a patient in need of such treatment, comprising
administering a therapeutically effective amount of the
pharmaceutical composition described above to said patient, thereby
treating said disease or disorder. Each possibility represents a
separate embodiment of the present invention.
[0130] In some embodiments, the method of treating the disease or
disorder may further include administering a second therapeutic
agent. In some embodiments, the second therapeutic agent may be a
PAR-1 antagonist.
[0131] There is provided a kit comprising a peptide conjugate for
the treatment of a disease or disorder associated with excessive
protease receptor activity, wherein the peptide conjugate may be as
set forth in formula I:
PRO-PEP-DIS (I)
wherein, PRO is a protecting moiety; PEP is a peptide moiety
comprising Aspartic acid-Proline-Arginine, or an active variant
thereof; and DIS is a protease-disabling moiety, and wherein PRO is
bound to the N'-terminus amino acid of PEP directly or via a
linker, and wherein DIS is bound to the C'-terminus amino acid of
PEP directly or via a linker.
[0132] In some embodiments, the kit may further include
instructions for use of the pharmaceutical composition for the
treatment of the disease or disorder. In some embodiments, the
instructions may further recite a web site address providing
further information, and troubleshooting among other services. In
some embodiments, the instructions may be on paper form such as a
package insert or label.
[0133] In some embodiments, the methods of the invention are aimed
at preventing excessive protease receptor activity, including,
preventing the onset of excessive protease receptor activity.
Determining the dose of the peptide conjugate required for
preventing protease receptor activity may be carried out by
assessing reduction in the PAR-1 levels following administration of
the peptide conjugate of the invention. Reduction in excessive
protease receptor activity refers to a decrease in, or an absence
of, activity of the receptor relative to the levels of activity
prior to administration or relative to a control value. Reduced
PAR-1 activity, includes reduction of activity by at least about
10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), as compared
to the level of activity just prior to administration. Hence, the
level of activity obtained from a sample of a patient in need
thereof may be compared to the level of PAR-1 activity in a sample
obtained from said patient in previous time points (e.g., prior to
administration of the peptide conjugate or during the onset of a
neuropathic disease or disorder). It is further optional to measure
PAR-1 activity in an individual prior to the onset of a disease or
disorder (e.g., during a regular check-up), in order to determine
the individual's baseline.
[0134] The PAR-1 activity in a patient in need thereof may be
compared to a standard or control obtained from normal individuals.
In one example, PAR-1 activity can be assessed in a population of
healthy individuals or individuals who have not had a neuropathic
disease or disorder. Such activity is referred to as a "negative
control." Conversely, PAR-1 may also be obtained from a pool of
individuals who are undergoing a neuroinflammation,
neuroinflammatory diseases or disorders, neurodegenerative disease
or disorder, neuropathy, or diabetes-related neuropathy, in order
to obtain a "positive control." Thus, in some embodiments,
following administration of the peptide conjugate of the invention,
the activity level of PAR-1 may decrease; the level(s) may get
closer to the level of the negative control, and farther from the
positive control. Alternatively, the PAR-1 levels of activity
decrease as compared to the levels during the onset of the
aforementioned disease or disorder.
[0135] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to". This term encompasses the terms "consisting of" and
"consisting essentially of". As used herein, the singular form "a",
"an", "the" and "said" include plural references unless the context
clearly dictates otherwise. For example, the term "a compound" or
"at least one compound" may include a plurality of compounds,
including mixtures thereof.
[0136] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the broad scope of the
invention. One skilled in the art can readily devise many
variations and modifications of the principles disclosed herein
without departing from the scope of the invention.
EXAMPLES
Example 1
Thrombin-Like Activity in Sciatic Nerves of Diabetic Rats
[0137] Thrombin-like activity was measured in sciatic nerves
derived from animal models of Guillain-Barre Syndrome (termed EAN,
for experimental autoimmune neuritis; FIG. 1A) and diabetes
(streptozotocine (STZ)-induced diabetes; FIG. 1B).
[0138] As presented in FIG. 1 the soluble thrombin-like activity
generated by, and released from, sciatic nerves of EAN or diabetic
(STZ) rats, into the surrounding medium, was compared to
thrombin-like activity in healthy control rats (n=12) and in CFA
rats (complete Freund's adjuvant). The latter control group refers
to rats treated with an inoculums containing the mycobacterium
tuberculosis emulsified in saline and CFA only. The results
indicate that thrombin-like activities were significantly elevated
in EAN rates 10 days after induction (Data is presented as a mean
thrombin like activity U/ml.+-.SEM, **=p<0.01;
*=p<0.005).
[0139] The results further indicate that thrombin-like activities
were significantly elevated in diabetic rats (FIG. 1B,
**=p=0.00071).
[0140] The conduction velocity of sciatic nerves derived from an
animal model of diabetes (streptozotocine (STZ)-induced diabetes;
FIG. 20) was measured in mice treated daily with sham (STZ), or
with the active drug TLDPR (peptide 5AA; SEQ ID NO: 2) nested
between N alpha-tosyl and chloromethyl ketone, namely TTN-ATLDPR-CK
(termed hereinafter T5AACK) injected in 100 microliter of 10 nM,
100 nM and 1 micromolar.
[0141] Conduction velocities in the sciatic nerves of the mice were
assessed by standard electrophysiology nerve conduction methods by
a blind assessor. The nerve conduction studies were carried out one
month after the induction of diabetes. A group of normal mice
without diabetes served as controls. All STZ treated mice developed
severe diabetes.
[0142] The comparison of sham treated and T5AACK 1 micromolar
treated mice was highly significant, resulting with p=0.00046
(T-test) and the effect of the 10 nM dose followed a similar trend,
resulting with p=0.016 (FIG. 20).
[0143] Thus, elevated thrombin-like activity in diabetic rats and
mice represents a potential target for intervention by the new
conjugates of the present invention.
Example 2
PAR-1 Expression Level in Sciatic Nerves of Diabetic Rats
[0144] The expression level of PAR-1 was measured in desheathed
sciatic nerves from STZ-induced diabetic rats and control
non-diabetic rats (FIG. 2A-2B).
[0145] Western blot analysis of PAR-1 level from sciatic nerves of
two STZ-induced rats, as compared to two healthy controls indicates
a significant decrease in PAR-1 level in STZ-induced rats
sciatic-nerves (n=3; p=0.012) compared to control non-diabetic rats
(FIG. 2B; data presented as a mean fold.+-.SEM, *=p<0.05).
[0146] Based on FIG. 2 and further data (not disclosed)
significantly low levels of PAR-1 are found in the animal models of
Guillain-Barre Syndrome (EAN) and diabetes (STZ-induced diabetic
rats). Without being bound by any theory or mechanism, such
findings may indicate an intrinsic protection mechanism employed by
the nerves to avoid PAR-1 over activation by the elevated
thrombin-like activities (such as presented in FIG. 1B). Thus,
PAR-1 sensitivity may strengthen the need to interfere with the
PAR-1 pathway and protect the receptor from over-activation.
Example 3
PN-1 Expression Level in Sciatic Nerves of Diabetic Rats
[0147] The expression level of PN-1 (thrombin-inhibitor, 143 kDa)
was measured in desheathed sciatic nerves from STZ-induced diabetic
rats and control non-diabetic rats, by Western blot analysis (FIGS.
3A-3B).
[0148] The result show a significant increase in PN-1 level in
STZ-induced rats sciatic-nerves (p=0.003) compared to control
non-diabetic rats (FIG. 3B; data presented as a mean fold.+-.SEM,
**=p<0.01).
[0149] Without being bound by any theory or mechanism, increased
levels of thrombin inhibitors, such as PN-1, in STZ-induced
diabetic rats is potentially an intrinsic protection mechanism
employed by the nerves to avoid PAR-1 over activation by the
elevated thrombin-like activities presented in FIG. 1. In addition,
the increased thrombin-inhibitor levels in diabetic rats may
indicate the need to regulate PAR-1 activating-proteases.
Example 4
Therapeutic Effect of Thrombin-Inhibitors on Electrophysiological
Parameter In Vivo
[0150] Electrophysiological tests of the sciatic nerves were
performed on day 32 PI (post-immunization/induction) in EAN model
(FIGS. 3A-C) and 8 weeks after STZ induction (FIG. 4A-C). Rats were
anesthetized with Phenobarbital (IP, 24 mg/kg). Two pairs of
monopolar needle electrodes were used to stimulate the tail nerves.
Stimulating cathodes were inserted to a depth of 4-5 mm at the base
of the tail and 4 cm distally. An anode at each location was
inserted into the skin (4-5 mm depth) 1 cm proximal to the cathode.
A ground electrode was placed between the distal stimulating
electrode and the active recording electrode. Electromyography
(EMG) recordings of responses of the tail muscles to proximal and
distal stimuli, made by a pair of ring electrodes coated with
electrode jelly and placed 1 cm distally to the distal stimulating
electrode, were collected. The tail skin was wiped with alcohol
prior to placing the electrodes. The EMG output was displayed on a
fully digital recording Keypoint apparatus (Dantec, Skovlunde,
Denmark). Both proximal and distal latencies were measured using
time intervals from the stimulus artifact to the first deflection
from baseline. To calculate the motor nerve conduction velocity
(MNCV), the distance between stimulating cathodes was divided by
the latency difference. Amplitudes of the compound muscle action
potential (CMAP) from both proximal and distal stimulations were
measured from the negative to the positive peaks. The reduction of
the proximal CMAP compared with that of the distal CMAP was
calculated by the equation: [(distal CMAP-proximal
CMAP)/distalCMAP.times.100] and was defined as the R ratio.
Temperature differences were minimized by conducting the study as
soon as the anesthesia had taken effect and by warming the tail
with a heating lamp.
[0151] Data was collected from EAN rats (FIG. 3) and STZ rats (FIG.
4) treated daily with the following thrombin-inhibitors: (i) N
alpha-tosyl-L-lysine chloromethyl ketone (TLCK) and (ii)
N.alpha.-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidin-
e (NAPAP), for 2 weeks.
[0152] FIGS. 3 and 4 each include 3 graphs summarizing the results
from electrophysiological tests of treated rats, as follows: distal
latency (3A, 4A) referring to the time, in milliseconds, between
distal stimulation to recording; conduction velocity (3B, 4B)
referring to the distance over time, in meters/seconds; and
amplitude (3C, 4C) referring to the size of the response as
measured in millivolts.
[0153] EAN and STZ rats which were not treated with TLCK or NAPAP
(FIG. 3B, `EAN` bar, and FIG. 4B, `STZ` bar) exhibited a
significantly reduced conduction velocity relative to control
(FIGS. 3B and 4B, black bars; *=p<0.05, **=p<0.01,
***=p<0.001). Conduction velocity in rats treated with TLCK was
significantly high compared to the saline treated EAN and STZ
control groups (*=p<0.05, **=p<0.01, ***=p<0.001;
STZ+TLCK: 41.95.+-.3.9 m/sec n=8 and STZ: 28.5.+-.2.8 m/sec n=4,
p=0.001 FIG. 3B, EAN+TLCK: 40.55.+-.6.7 m/sec n=6 EAN: 23.6.+-.1.2
m/sec n=5, p=0.05). NAPAP treatment group showed some improvement
in conduction velocities compared to the unaffected control group
(EAN+NAPAP: 36.05.+-.3.57 m/sec, n=5 and EAN: 23.6.+-.1.2 m/sec,
n=5, p=0.003, FIG. 3B; STZ+NAPAP: 40.1.+-.2.7 m/sec n=5 and STZ:
28.5.+-.2.8 m/sec n=5, p=0.01, FIG. 4B). TLCK treatment increased
proximal amplitude (EAN 0.76.+-.0.33, EAN+NAPAP 0.83.+-.0.4,
EAN+TLCK 1.36.+-.0.49), in a non-significant manner (FIG. 3B). The
beneficial effect on the proximal amplitude observed in the STZ
model (FIG. 4C) was also insignificant: STZ+TLCK 1.81.+-.0.8 (n=8),
STZ+NAPAP 1.68.+-.0.64 (n=5) compared to STZ: 0.48.+-.0.13
(n=5).
[0154] Latency, the time between induction and nerve conduction,
was also measured. The results indicate that in the STZ group an
increased latency was 1.93.+-.0.139 msec (n=5) compared to control
(1.29.+-.0.057 msec, n=4). The NAPAP and TLCK treatments caused
decreased latencies of 1.676.+-.0.132 msec (n=5, p=0.11) and
1.44.+-.0.04 msec (n=5, p=0.0019), respectively. In the EAN group
an increased latency was detected (1.43.+-.0.023 msec, n=5)
compared to control (0.54.+-.0.03 msec, n=3). The NAPAP and TLCK
treatments did not caused decreased latencies (1.32.+-.0.33 msec,
n=5, p=0.39, 1.28.+-.0.18 msec, n=5, p=0.27, respectively).
[0155] The results indicate that treatment with thrombin inhibitors
improves nerve conduction in an animal model for diabetes. This
outcome was however accompanied by internal bleeding.
Example 5
PAR-1 Expression Level Ex-Vivo in Brain Homogenates of an Animal
Model for ALS (SOD-1 Mice)
[0156] The expression level of PAR-1 was measured in brain
homogenates derived from SOD-1 and wild type control mice.
[0157] FIG. 5 depicts a Western blots (A) and the corresponding
analysis, performed with ImageJ software, from several independent
experiments (B), of PAR-1 expression levels in brain homogenates of
four SOD-1 mice, as compared to five healthy controls. The results
demonstrate a significant decrease (2.8 fold decrease) in the
expression of PAR-1 in SOD-1 mice compared to wild-type controls
(p<0.001). The results may indicate that interfering with the
PAR-1 pathway and protecting PAR-1 from over-activation would be
beneficial in treating neuropathic diseases and disorders.
Example 6
Survival Experiment of ALS (SOD-1) Mice Treated with General
Thrombin Inhibitors
[0158] ALS (SOD-1) mice were randomly divided into three groups,
and were either untreated (diamond), treated with a relatively
general thrombin inhibitor, N alpha-tosyl-L-lysine chloromethyl
ketone (4.4 mg/kg TLCK; circle-), or treated with a PAR-1
antagonist (25 .mu.g/kg SCH79797; triangles).
[0159] The results indicate that survival without treatment did not
last more than 145 days. However, treatment with either PAR-1
antagonist or with a thrombin inhibitor improved survival by at
least 15 days (FIG. 6). Since PAR-1 is decreased as the disease
progresses, utilizing a specific PAR-1-based thrombin inhibitor in
advanced stages of the disease seems more reasonable than utilizing
a PAR-1 antagonist.
Example 7
Thrombin-Like Activity in Glioma Cell Lines
[0160] Thrombin-like activity was measured in C6 and CNS-1 glioma
cell-lines and their surrounding medium (FIGS. 7A and 7B,
respectively), four hours after cells were treated with the highly
selective thrombin inhibitor
N.alpha.-(2-naphthyl-sulphonyl-glycyl)-DL-p-amidinophenylalanyl-
-piperidine, NAPAP (FIGS. 7A-7B, grey bars). Results are
demonstrated relative to control, non-treated, cells (FIGS. 7A-7B,
black bars).
[0161] It is evident from FIG. 7 that (a) thrombin-like activity
was restricted to the cells themselves, with no apparent activity
in the surrounding medium, and (b) thrombin-like activity was
inhibited by NAPAP in both cell lines. Since thrombin is known to
increase the proliferation of glioma cells, the glioma cell-lines
endogenous generation of thrombin-like activity indicates the
potential beneficial effect of thrombin specific inhibitor as
proliferation regulators in gliomas such as Glioblastoma multiform
(GBM).
Example 8
Proliferation-Inhibition of Glioma Cell Lines by a PAR-1
Antagonist
[0162] Several peptide moieties (Table 1) according to the present
invention were synthesized. Proliferation was measured, by means of
XTT, in C6 and CNS-1 glioma cell-lines (FIGS. 8A and 8B,
respectively). In C6 cells, proliferation was measured before
(black bar) and after cells were treated with the PAR-1 antagonist
SCH79797 at various doses ranging from 10 nM to 10 .mu.M (grey
bars; FIG. 8A). In CNS-1 cells, proliferation was measured before
(black bar) and after cells were treated with the PAR-1 antagonist
SCH79797 (1 .mu.M), the thrombin inhibitor NAPAP, the thrombin
inhibitor TLCK, or combinations thereof (grey bars; FIG. 8B).
[0163] It is evident from FIG. 8A that the PAR-1 antagonist
inhibited proliferation in a dose-dependent manner in C6 cells.
Furthermore, according to the results presented in FIG. 8B, NAPAP
(1 .mu.M) or TLCK (1 .mu.M) did not inhibit proliferation of CNS-1
cells when administered alone. However, combining NAPAP or TLCK
with a PAR-1 antagonist seems to improve the inhibitory effect of
the PAR-1 antagonist.
TABLE-US-00001 TABLE 1 Amino-acid sequences of peptide moieties.
SEQ ID Amino-acid sequence Peptide Name -- DPR 3AA SEQ ID NO: 1
LDPR 4AA SEQ ID NO: 2 TLDPR 5AA SEQ ID NO: 3 ATLDPR 6AA SEQ ID NO:
4 NATLDPR 7AA SEQ ID NO: 5 TNATLDPR 8AA SEQ ID NO: 6 ATNATLDPR 9AA
SEQ ID NO: 7 KATNATLDPR 10AA SEQ ID NO: 8 SKATNATLDPR 11AA SEQ ID
NO: 9 ESKATNATLDPR 12AA SEQ ID NO: 10 PESKATNATLDPR Segment of the
binding site on the receptor PAR-1 SEQ ID NO: 11 RPESKATNATLDPR
14AA SEQ ID NO: 12 RRPESKATNATLDPR 15AA SEQ ID NO: 13
ARRPESKATNATLDPR 16AA SEQ ID NO: 14 RARRPESKATNATLDPR 17AA SEQ ID
NO: 15 TRARRPESKATNATLDPR 18AA SEQ ID NO: 16 RTRARRPESKATNATLDPR
19AA SEQ ID NO: 17 ARTRARRPESKATNATLDPR C' terminus segment of
PAR-1 SEQ ID NO: 18 LDPRSFLLRNPNDKYEPF amino-terminal exodomain of
PAR-1 SEQ ID NO: 19 KYEPF hirudin-like motif
[0164] The peptide moieties were derived from the C'-terminus of
PAR-1. A comparison between the amino acid (AA) sequence upstream
and downstream to thrombin cleavage-site in human, mouse, rat and
bovine indicates that these sequences are by and large conserved
(Table 2). The position of the amino acids corresponding to the
thrombin cleavage site is indicated for each sequence in the second
column of Table 2 and the designed peptide length is indicated in
the third column of this Table. Positions 35-47 represent part of
the N-terminal of PAR-1, also termed P1-P7, designating the
position upstream to the cleavage site.
TABLE-US-00002 TABLE 2 PAR-1 amino-acids sequence upstream and
downstream to thrombin cleavage-site in human, mouse, rat and
bovine. P P P P P P P Thrombin 7 6 5 4 3 2 1 cleavage 3 3 3 3 3 4 4
4 4 4 4 4 4 Species site Position 5 6 7 8 9 0 1 2 3 4 5 6 7 Human
41-42 PEPTIDE N A T L D P R S F 3AA D P R 4AA L D P R 5AA T L D P R
6AA A T L D P R 7AA N A T L D P R Mouse 41-42 PEPTIDE D A T V N P R
S F 3AA N P R 4AA V N P R 5SAA T V N P R 6AA A T V N P R 7AA D A T
V N P R Rat 45-46 PEPTIDE Y A T P N P R S F 3AA N P R 4AA P N P R
5AA T P N P R 6AA A T P N P R 7AA Y A T P N P R Bovine 41-42
PEPTIDE N G T L G P R S F F L R N 3AA G P R 4AA L G P R 5AA T L G P
R 6AA G T L G P R 7AA N G T L G P R
[0165] Furthermore, PAR-1 antagonist (1 .mu.M) alone caused a
significant 25% proliferation-inhibition in CNS-1 cells, wherein a
combined treatment of the PAR-1 antagonist with NAPAP or TLCK
significantly increased said inhibitory effect. The decreased
proliferation of glioma cells in response to PAR-1 pathway
modulation is a major finding, suggestive of the potential of
PAR-1-based compounds as thrombin inhibitor for treating neuropathy
and glioma, such as, GBM.
[0166] In another set of experiments, CNS-1 glioma cell-line was
treated with increased doses of TLCK (125 nM to 14 mM), for 48
hours (FIG. 9; grey bars). Proliferation was measured by means of
XTT in comparison to non-treated cells (control; black bar). High
concentrations of TLCK (6.7 mM, 14 mM) caused cell death, and at
the micro-molar range (8-33 .mu.M) a significant
proliferation-inhibition occurred. No significant
proliferation-inhibition was seen between 1 .mu.M to 125 nM
[0167] It is therefore evident that the highly effective thrombin
inhibitor TLCK decreased glioma-cell proliferation at relatively
high concentrations (i.e. higher than 1 .mu.M), with the downside
of increased risk for hemorrhage. The new PAR-1-based conjugates
provided by the present invention provide the required activity at
significantly lower concentrations and are devoid of harmful side
effects, as demonstrated in the following Examples.
Example 9
Inhibition of Cell Proliferation
[0168] Initially, CNS-1 glioma cells were treated with increased
concentrations of two peptide conjugates: a short conjugate of the
formula tosyl-DPR-CK (comprising 3 amino acids, 3AA) or a long
conjugate of the formula tosyl-NATLDPR-CK (comprising 7 amino
acids, 7AA; SEQ ID NO: 4) in concentrations of 100 pM, 1 nM and 10
nM, for 48 hours (FIG. 10). FIG. 10 provides the results of an XTT
assay used for assessing cell proliferation. The results indicate
that the short conjugate (3AA) caused significant inhibition of
cell proliferation at 1 nM and higher inhibition at 10 nM, while
the long conjugate (7AA) caused a significant inhibition at 10 nM,
relative to untreated control (FIG. 10).
[0169] CNS-1 glioma cells were treated with increased
concentrations of the peptide conjugates containing the peptide DPR
or the peptides of SEQ ID NOs: 2-4 for 48 hours and for 72 hours
(FIGS. 11 and 12, respectively). As seen in FIG. 11, a trend to
reduced proliferation was observed in the samples treated with the
peptide conjugates compared to controls at 48 hours. Significant
inhibition, relative to untreated controls, was noted for 10 .mu.M
peptide conjugate encompassing the peptides 6AA and 7AA
(p<0.05). At the 72 hour experiment (FIG. 12) significantly
lower proliferation was found for all concentrations of the peptide
conjugate containing the 6AA peptide compared to controls
(p<0.05). This observation was most significant at
concentrations of 1 and 10 micromolar (p=0.002 and 0.003,
respectively). Similar inhibitory effect was observed under
treatment with the peptide conjugate that includes the 3AA peptide,
at all tested concentrations, compared to controls (p<0.05). The
results indicate that the inhibition of cell proliferation as
exhibited by the peptide conjugates is significantly greater than
cell proliferation of the control cells, namely, cells not
incubated with any of the peptide conjugates. Moreover, the
inhibitory effect exerted by the peptide conjugate was prominent
even at concentrations as low as the range of micromolars and
nanomolars.
Example 10
Proliferation Inhibition Relative to PAR-1 Inhibitory Activity
[0170] The effect of the peptide conjugate alone, or in combination
with PAR-1 antagonist, SCH79797, on cell proliferation was
determined in comparison to untreated cells or cells treated with
PAR-1 antagonist (FIG. 13). The results indicate that the peptide
conjugate, as exemplified by a conjugate comprising the peptide set
forth in SEQ ID NO:4, induces a greater inhibition on cell
proliferation when applied alone or in combination with PAR-1
antagonist, compared to control (untreated cells) and even compared
to PAR-1 antagonist alone.
Example 11
Anti-Thrombin-Activity Ex-Vivo
[0171] The activity of high concentrations (0.05 U/ml) of thrombin
(FIGS. 14-14B; diamond) was measured after treatment with a wide
dose-range of either the short conjugate (FIG. 14A, tosyl-DPR-CK,
3AA) or the long conjugate (FIG. 14B, tosyl-NATLDPR-CK, 7AA).
Measurements were carried out in a thrombin inhibition non-cellular
assay, which included a buffer, thrombin and a specific thrombin
substrate which its cleavage indicates thrombin activity or
inhibition. The conjugates concentrations applied were as follows:
control (no treatment--0 nM; full diamond), 1 mM (vertical line),
50 .mu.M (circle), 10 .mu.M (cross), 1.25 .mu.M (square) and 156 nM
(triangle).
[0172] The data presented in FIGS. 14A-14B demonstrate that both
peptide-conjugates have the ability to exert a complete inhibition
of thrombin activity at a wide range of concentration, ranging from
as high as 1 mM to as low as 156 nM.
[0173] FIG. 15 presents thrombin activity in the presence of
peptide conjugates containing the peptide DPR (also termed 3AA;
FIG. 15: x symbols on grey line) or the peptides set forth in SEQ
ID NOs: 1-4 (also termed 4AA-7AA, respectively; FIG. 15: 4AA-x
symbols on dark line, 5AA-triangles, 6AA-squares, 7AA-diamonds)
determined in a non-cellular system as described above. The
corresponding IC50 of peptide conjugates are listed in Table 3
below. These results demonstrate the efficacy of the peptide
conjugates in inhibiting bovine serum thrombin.
TABLE-US-00003 TABLE 3 IC50 of the peptide conjugates Peptide
encompassed in the conjugate IC50 (SEQ ID NO.) 700 pM 3AA --
.sup..sctn.4AA (1).sup. 700 pM 5AA (2) 700 pM 6AA (3) 100 pM 7AA
(4) .sup..sctn.Results were inconsistent
Example 12
Anti-Thrombin-Activity in CNS-1 Cells
[0174] The thrombin-like activity of CNS-1 cells was initially
measured in control cells and in cells treated with (i) the short
peptide conjugate (tosyl-DPR-CK, corresponding to a conjugate that
includes the peptide termed 3AA) or (ii) the long conjugate
(tosyl-NATLDPR-CK, corresponding to a conjugate that includes the
peptide termed 7AA (SEQ ID No. 4)) at high (10 mM) and low (100 nM)
concentrations (FIG. 16).
[0175] At all range of concentrations, the peptide-conjugates
induced a significant inhibition of thrombin-like activity
generated by CNS-1 glioma cells.
[0176] FIG. 17 presents thrombin activity in the presence of
peptide conjugates containing the peptide DPR (also termed 3AA;
FIG. 17: asterisk) or the peptides set forth in SEQ ID NOs: 1-4
(also termed 4AA-7AA, respectively; FIG. 17: 4AA-x, 5AA-triangle,
6AA-square, 7AA-diamond) determined in rat glioma cells as
described above. The corresponding IC50 of peptide conjugates are
listed in Table 4 below:
TABLE-US-00004 TABLE 4 IC50 of the peptide conjugates in rat glioma
cells Peptide encompassed in the conjugate IC50 (SEQ ID NO.) 7 pM
3AA -- 4AA (1) 700 pM 5AA (2) 700 fM.sup. 6AA (3) 10 pM 7AA (4)
Example 13
Plasma Concentrations of the Conjugates In-Vivo
[0177] The concentrations of the peptide conjugates in the plasma
of animals treated therewith was evaluated by a unique method that
was based on the ability of the peptide conjugates to inhibit
thrombin activity. Data was obtained for conjugate peptides
comprising the peptide DPR and the peptides set forth in SEQ ID NO:
2; SEQ ID NO: 3; and SEQ ID NO: 4 (FIGS. 18A-18D, respectively).
The essay initiated by obtaining blood samples from animals which
received the peptide conjugates (100 microliter containing 2
micromolar, i.p.). The blood samples, after being maintained in
citrate buffered sample tubes, were subjected to centrifugation for
plasma separation. Samples were then filtered through a membrane
with a 10 kD molecular weight cutoff which excluded all blood
coagulation factors (by addition of calcium to neutralize the
anticoagulation effect of the citrate buffer). Coagulation
factor-free plasma samples were then applied to the thrombin
activity assay.
[0178] The resulting thrombin activities in each plasma sample
either diluted (.times.10) or as is were given in arbitrary units
(FIGS. 18-18D, diagonal shading and dotted bars, respectively).
However, the actual concentration were deduced by comparing the
activities to calibrated activities generated with known
concentrations of the conjugate peptides. The calibration data
included the following thrombin activities: activity in a sample of
thrombin in the absence of plasma and conjugates (black bars, FIGS.
18A-18D); and activity in four samples of thrombin and known
concentrations of the peptide conjugates (10 nM, 100 nM, 1
micromolar and 10 micromolar; bars 2.sup.nd to 5th from right in
FIGS. 18A-18D). The comparison to calibration data indicates that
an i.p. injection of 100 .mu.l containing 2 micromolar of each
peptide conjugate resulted in a serum concentration of about 1
.mu.M for the peptide conjugate containing the 3AA peptide, less
than 10 nM for the peptide conjugates containing the 5AA and 6AA
peptides, and about 10 nM for the peptide conjugates containing the
7AA peptide. The results also suggest that the assay used herein is
suitable for measuring the levels of the peptide conjugate and to
identify significant differences in their distribution and
stability in vivo.
Example 14
The Effect of the Peptide Conjugates on Blood Coagulation
[0179] Tables 5-9 below represent the effect of the peptide
conjugates comprising the peptide DPR and the peptides set forth in
SEQ ID NOs: 1-4 on the following standard blood coagulation tests:
Prothrombin time (PT, Table 5), activated Partial thromboplastin
time (aPTT, Table 6), thrombin time (TT, Table 7), Factor Xa
activity (Fxa; Table 8) and Protein C activity (Table 9) using
standard hematology lab tests. For each test the most potent
peptide conjugate to least potent were scored from (1) to (5). In
the PT and PTT tests only very high concentrations (100 .mu.M) of
the peptide conjugate significantly affected coagulation. Since a
selection towards the conjugates least likely to affect coagulation
systemically was applied, the peptide conjugate having the 3AA
peptide stood out as having significantly more deleterious effects
on coagulation. The most sensitive test for verifying the effect of
the peptide conjugates in general was TT. Without being bound to
any theory or mechanism, it is assumed that the TT test was most
sensitive because it supports the mechanism of action of the
peptide conjugates as direct thrombin inhibitors. Thus, the TT test
indicates that the conjugates are useful inhibitors. Moreover, the
results suggest that the peptide conjugate exert their therapeutic
activity without interfering with coagulation. This is an
outstanding benefit, as the current thrombin inhibitors cause
internal bleeding.
TABLE-US-00005 TABLE 5 Prothrombin time test PT 0 10 nM 1 .mu.M 100
.mu.M (Potency) 3AA 11.5 11.5 12.3 FAILED (1) 4AA 11.5 11.5 11.4
12.7 (5) 5AA 11.5 12.1 11.5 14.5 (4) 6AA 11.5 11.5 11.4 16.5 (3)
7AA 11.5 11.5 11.2 17.4 (2)
TABLE-US-00006 TABLE 6 Activated Partial thromboplastin time test
aPTT 0 10 nM 1 .mu.M 100 .mu.M (Potency) 3AA 30.5 30 38.1 150 (1)
4AA 30.5 30.8 30.9 42.6 (5) 5AA 30.5 30.1 30.5 63.9 (4) 6AA 30.5 30
30.6 67.6 (3) 7AA 30.5 29.9 30.7 85.2 (2)
TABLE-US-00007 TABLE 7 Thrombin time test TT 0 10 nM 1 .mu.M
(Potency) 100 .mu.M 3AA 13.4 14.1 >80 (1) >80 4AA 13.4 13.5
14.6 (5) >80 5AA 13.4 13.8 17.8 (4) >80 6AA 13.4 13.9 18.5
(3) >80 7AA 13.4 13.5 24.1 (2) >80
TABLE-US-00008 TABLE 8 Factor Xa activity test Fxa 0 1 .mu.M 100
.mu.M (Potency) 3AA 0.11 0.21 FAILED (1) 4AA 0.11 0.11 (5) 5AA 0.11
0.13 (4) 6AA 0.11 0.36 (3) 7AA 0.11 0.43 (2)
TABLE-US-00009 TABLE 9 Protein C activity test aPC 0 1 .mu.M 100
.mu.M (Potency) 3AA 109 107 FAILED (1) 4AA 109 108 66 (5) 5AA 109
109 20 (3) 6AA 109 108 11 (2) 7AA 109 110 25 (4)
Example 15
Conduction Velocities in the Sciatic Nerve of an Animal Model for
Diabetic Neuropathy
[0180] Streptozotocyn (STZ) was dissolved in citrate buffer pH 4.5
and a single dose of STZ was administered IP (140 mg/kg) to 12
weeks BALB/c mice. Hyperglycemia (over 300 mg/dl blood glucose) was
observed in 90% of the mice in 3 repeated measurements within a
week from the STZ injection. Mice developed diabetic neuropathy
developed within 4 weeks after the induction of diabetes. Mice were
treated daily with sham (STZ), or with the a peptide conjugate as
the active drug, exemplified by the conjugate T5AACK (i.e. a
conjugate comprising the peptide set forth in SEQ ID NO:2) injected
in 100 microliter volume containing the following doses: 10 nM, 100
nM and 1 micromolar. A group of normal mice without diabetes served
as controls.
[0181] Conduction velocities in the sciatic nerves of the mice were
assessed by standard electrophysiology nerve conduction methods by
a blind assessor (FIG. 19). T-test analysis of indicated that the
effect of the peptide conjugate T5AACK relative to sham treated
mice was highly significant: 1 micromolar, p=0.00046 and 10 nM,
p=0.016.
[0182] Thus, the conduction velocity of the sciatic nerve in
diabetic mice treated with the peptide conjugate resembled that of
normal, health mice.
Example 16
Tumor Size and Edema in GBM Animal Model
[0183] The therapeutic effect of the peptide conjugate was
determined in-vivo in an animal (rat) model of GBM using a peptide
conjugate that includes the 6AA peptide (SEQ ID NO: 3).
[0184] Cells (0.5.times.10.sup.6 cells) from a highly malignant
cell line of rat Glioblastoma multiforme (GBM) were injected by
stereotactic methods into the parietal cortex of rats. Five (5)
days later the brain of these rats were examined by MM and the
tumors scored on a scale of 1-3 (1-small size tumor, 2-middle size
tumor, 3-large size tumor) and for ventricular localization of the
tumor. A catheter was placed in the brain tumor and attached to an
osmotic pump (Alzet) releasing 1 .mu.l/h for 7 days of one of the
following three (3) treatments: saline (n=13), 6AA 2 .mu.M (n=14)
and 6AA 20 .mu.M (n=14). Following seven (7) days of treatments an
MRI was performed and the size of the tumor and edema surrounding
the tumor was analyzed by standard ROI manual techniques for each
rat and summarized for all three groups of treatment (1) control;
(2) treatment with 2 .mu.M of a peptide conjugate comprising the
peptide termed 6AA (SEQ ID NO: 3); and (3) treatment with 20 .mu.M
of a peptide conjugate comprising the peptide termed 6AA (SEQ ID
NO: 3).
[0185] In order to account for the unequal distribution of initial
tumor size, tumor and edema analyses were performed on rats with
grade 1 and 2 tumors in the initial Mill (n=7, 6 and 5 for the
saline, low and high treatment groups, respectively). The results
for the changes in tumor size (i.e. the size obtained in second
(post treatment) MM minus the size in initial (baseline, t=0) Mill)
and similarly change in edema are presented in FIGS. 20 and 21,
respectively. As shown by FIGS. 20 and 21, high dose treatment
reduced tumor size to half of the original size (p=0.030, t-test).
A similar trend was observed for the low dose group. In addition,
both doses caused reduction in the edema surrounding the
tumors.
[0186] Survival was registered daily (FIG. 21). None of the 3 rats
with severe initial tumors (grade 3 or ventricular) survived to
perform the second Mill, compared to all 3 severe tumor rats in the
high dose (6AA 20 .mu.M) treatment group and 1 out of 3 in the low
dose group (p=0.05 for the high dose group compared to controls and
p=0.024 for the high dose group compared to the other 2). As shown
in FIG. 21, there was a clear trend to increased survival in the
high dose 20 .mu.M group and this group included 2 mice with
exceptionally long survival which is borderline in significance to
no survival in the other groups (p=0.055 by Fisher's exact
test).
[0187] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the invention.
Sequence CWU 1
1
1914PRTArtificial sequencePeptide 1Leu Asp Pro Arg 1
25PRTArtificial sequencePeptide 2Thr Leu Asp Pro Arg 1 5
36PRTArtificial sequencePeptide 3Ala Thr Leu Asp Pro Arg 1 5
47PRTArtificial sequencePeptide 4Asn Ala Thr Leu Asp Pro Arg 1 5
58PRTArtificial sequencePeptide 5Thr Asn Ala Thr Leu Asp Pro Arg 1
5 69PRTArtificial sequencePeptide 6Ala Thr Asn Ala Thr Leu Asp Pro
Arg 1 5 710PRTArtificial sequencePeptide 7Lys Ala Thr Asn Ala Thr
Leu Asp Pro Arg 1 5 10 811PRTArtificial sequencePeptide 8Ser Lys
Ala Thr Asn Ala Thr Leu Asp Pro Arg 1 5 10 912PRTArtificial
sequencePeptide 9Glu Ser Lys Ala Thr Asn Ala Thr Leu Asp Pro Arg 1
5 10 1013PRTArtificial sequencePeptide 10Pro Glu Ser Lys Ala Thr
Asn Ala Thr Leu Asp Pro Arg 1 5 10 1114PRTArtificial
sequencePeptide 11Arg Pro Glu Ser Lys Ala Thr Asn Ala Thr Leu Asp
Pro Arg 1 5 10 1215PRTArtificial sequencePeptide 12Arg Arg Pro Glu
Ser Lys Ala Thr Asn Ala Thr Leu Asp Pro Arg 1 5 10 15
1316PRTArtificial sequencePeptide 13Ala Arg Arg Pro Glu Ser Lys Ala
Thr Asn Ala Thr Leu Asp Pro Arg 1 5 10 15 1417PRTArtificial
sequencePeptide 14Arg Ala Arg Arg Pro Glu Ser Lys Ala Thr Asn Ala
Thr Leu Asp Pro 1 5 10 15 Arg 1518PRTArtificial sequencePeptide
15Thr Arg Ala Arg Arg Pro Glu Ser Lys Ala Thr Asn Ala Thr Leu Asp 1
5 10 15 Pro Arg 1619PRTArtificial sequencePeptide 16Arg Thr Arg Ala
Arg Arg Pro Glu Ser Lys Ala Thr Asn Ala Thr Leu 1 5 10 15 Asp Pro
Arg 1718PRTArtificial sequencePeptide 17Leu Asp Pro Arg Ser Phe Leu
Leu Arg Asn Pro Asn Asp Lys Tyr Glu 1 5 10 15 Pro Phe
1818PRTArtificial sequencePeptide 18Leu Asp Pro Arg Ser Phe Leu Leu
Arg Asn Pro Asn Asp Lys Tyr Glu 1 5 10 15 Pro Phe 195PRTArtificial
sequencePeptide 19Lys Tyr Glu Pro Phe 1 5
* * * * *